The present invention relates to methods and compositions for the expression and the secretion of biologically active polypeptides from genetically engineered duckweed.
The duckweeds are the sole members of the monocotyledonous family Lemnaceae. The four genera and 34 species are all small, free-floating, fresh-water plants whose geographical range spans the entire globe (Landolt (1986) Biosystematic Investigation on the Family of Duckweeds: The Family of Lemnaceaexe2x80x94A Monograph Study Geobatanischen Institut ETH, Stiftung Rubel, Zurich). Although the most morphologically reduced plants known, most duckweed species have all the tissues and organs of much larger plants, including roots, stems, flowers, seeds and fronds. Duckweed species have been studied extensively and a substantial literature exists detailing their ecology, systematics, life-cycle, metabolism, disease and pest susceptibility, their reproductive biology, genetic structure, and cell biology. (Hillman (1961) Bot. Review 27:221; Landolt (1986) Biosystematic Investigation on the Family of Duckweeds: The Family of Lemnaceaexe2x80x94A Monograph Study Geobatanischen Institut ETH, Stiftung Rubel, Zurich).
The growth habit of the duckweeds is ideal for microbial culturing methods. The plant rapidly proliferates through vegetative budding of new fronds, in a macroscopic manner analogous to asexual propagation in yeast. This proliferation occurs by vegetative budding from meristematic cells. The meristematic region is small and is found on the ventral surface of the frond. Meristematic cells lie in two pockets, one on each side of the frond midvein. The small midvein region is also the site from which the root originates and the stem arises that connects each frond to its mother frond. The meristematic pocket is protected by a tissue flap. Fronds bud alternately from these pockets. Doubling times vary by species and are as short as 20-24 hours (Landolt (1957) Ber. Schweiz. Bot. Ges. 67:271; Chang et al. (1977) Bull. Inst. Chem. Acad. Sin. 24:19; Datko and Mudd (1970) Plant Physiol. 65:16; Venkataraman et al. (1970) Z. Pflanzenphysiol. 62:316).
Intensive culture of duckweed results in the highest rates of biomass accumulation per unit time (Landolt and Kandeler (1987) The Family of Lemnaceaexe2x80x94A Monographic Study Vol. 2: Phytochemistry, Physiology, Application, Bibliography, Veroffentlichungen des Geobotanischen Institutes ETH, Stiftung Rubel, Zurich), with dry weight accumulation ranging from 6-15% of fresh weight (Tillberg et al. (1979) Physiol. Plant. 46:5; Landolt (1957) Ber. Schweiz. Bot. Ges. 67:271; Stomp, unpublished data). Protein content of a number of duckweed species grown under varying conditions has been reported to range from 15-45% dry weight (Chang et al (1977) Bull. Inst. Chem. Acad. Sin. 24:19; Chang and Chui (1978) Z. Pflanzenphysiol. 89:91; Porath et al. (1979) Aquatic Botany 7:272; Appenroth et al. (1982) Biochem. Physiol. Pflanz. 177:251). Using these values, the level of protein production per liter of medium in duckweed is on the same order of magnitude as yeast gene expression systems.
Sexual reproduction in duckweed is controlled by medium components and culturing conditions, including photoperiod and culture density. Flower induction is a routine laboratory procedure with some species. Plants normally self-pollinate, and selfing can be accomplished in the laboratory by gently shaking cultures. By this method, inbred lines of Lemna gibba have been developed. Spontaneous mutations have been identified (Slovin and Cohen (1988) Plant Physiol. 86, 522), and chemical and gamma ray mutagenesis (using EMS or NMU) have been used to produce mutants with defined characteristics. Outcrossing of L. gibba is tedious but can be done by controlled, hand pollination. The genome size of the duckweeds varies from 0.25-1.63 pg DNA/2C with chromosome counts ranging from 20 to 80 and averaging about 40 across the Lemnaceae (Landolt (1986) Biosystematic Investigation on the Family of Duckweeds: The family of Lemnaceaexe2x80x94A Monograph Study Geobatanischen Institut ETH, Stiftung Rubel, Zurich). Ploidy levels are estimated to range from 2-12 C. Id. Genetic diversity within the Lemnaceae has been investigated using secondary products, isozymes, and DNA sequences (McClure and Alston (1966) Nature 4916:311; McClure and Alston (1966) Amer. J. Bot. 53:849; Vasseur et al. (1991) Pl. Syst. Evol. 177:139 (1991); Crawford and Landolt (1993) Syst. Bot. 10:389).
Duckweed plant or duckweed nodule cultures can be efficiently transformed with an expression cassette containing a nucleotide sequence of interest by any one of a number of methods including Agrobacterium-mediated gene transfer, ballistic bombardment, or electroporation. Stable duckweed transformants can be isolated by transforming the duckweed cells with both the nucleotide sequence of interest and a gene which confers resistance to a selection agent, followed by culturing the transformed cells in a medium containing the selection agent. See U.S. Pat. No. 6,040,498 to Stomp et al.
A duckweed gene expression system provides the pivotal technology that would be useful for a number of research and commercial applications. For plant molecular biology research as a whole, a differentiated plant system that can be manipulated with the laboratory convenience of yeast provides a very fast system in which to analyze the developmental and physiological roles of isolated genes. For commercial production of valuable polypeptides, a duckweed-based system has a number of advantages over existing microbial or cell culture systems. Plants demonstrate post-translational processing that is similar to mammalian cells, overcoming one major problem associated with the microbial cell production of biologically active mammalian polypeptides, and it has been shown by others (Hiatt (1990) Nature 334:469) that plant systems have the ability to assemble multi-subunit proteins, an ability often lacking in microbial systems. Scale-up of duckweed biomass to levels necessary for commercial production of recombinant proteins is faster and more cost efficient than similar scale-up of mammalian cells, and unlike other suggested plant production systems, e.g., soybeans and tobacco, duckweed can be grown in fully contained and controlled biomass production vessels, making the system""s integration into existing protein production industrial infrastructure far easier.
These characteristics make duckweed an ideal choice to develop as an efficient, plant-based system for the production of recombinant proteins. Accordingly, the present invention provides methods and compositions that increase the efficiency of the duckweed gene expression system as a tool for producing biologically active polypeptides.
The present invention is drawn to methods and compositions for the expression and recovery of biologically active recombinant polypeptides, using duckweed as the expression system. One aspect of the present invention provides a method for enhanced expression levels of biologically active polypeptides in duckweed, resulting in an increased polypeptide yield and enabling the production of useful quantities of valuable biologically active polypeptides in this system. Another aspect of the invention discloses methods for the directed secretion of biologically active polypeptides from genetically engineered duckweed plant or duckweed nodule cultures. Secretion of the expressed polypeptide facilitates its recovery and prevents the loss of activity that might result from the mechanical grinding or enzymatic lysing of the duckweed tissue.
In one embodiment, the invention encompasses a method of producing a biologically active recombinant polypeptide in a duckweed plant culture or a duckweed nodule culture, comprising the steps of: (a) culturing within a duckweed culture medium a duckweed plant culture or a duckweed nodule culture, wherein said duckweed plant culture or said duckweed nodule culture is stably transformed to express said biologically active recombinant polypeptide, and wherein said biologically active recombinant polypeptide is expressed from a nucleotide sequence comprising a coding sequence for the polypeptide and an operably linked coding sequence for a signal peptide that directs secretion of the polypeptide into the culture medium; and (b) collecting said biologically active polypeptide from the duckweed culture medium. In some embodiments of this method, the nucleotide sequence has at least one attribute selected from the group consisting of: (a) duckweed-preferred codons in the coding sequence for said polypeptide; (b) duckweed-preferred codons in the coding sequence for said signal peptide; (c) a translation initiation codon that is flanked by a plant-preferred translation initiation context nucleotide sequence; and (d) an operably linked nucleotide sequence comprising a plant intron that is inserted upstream of the coding sequence. In some embodiments of this method, the biologically active recombinant polypeptide is secreted into the duckweed culture medium.
In another embodiment, the invention encompasses a method of secreting a biologically active recombinant polypeptide in a duckweed plant culture or a duckweed nodule culture, comprising the steps of: (a) culturing within a duckweed culture medium a duckweed plant culture or a duckweed nodule culture, wherein said duckweed plant culture or said duckweed nodule culture is stably transformed to express said biologically active recombinant polypeptide, and wherein said biologically active recombinant polypeptide is expressed from a nucleotide sequence comprising a coding sequence for the polypeptide and an operably linked coding sequence for a signal peptide that directs secretion of the polypeptide into the culture medium; and (b) collecting said biologically active polypeptide from the duckweed culture medium. In some embodiments of this method, the nucleotide sequence has at least one attribute selected from the group consisting of: (a) duckweed-preferred codons in the coding sequence for said polypeptide; (b) duckweed-preferred codons in the coding sequence for said signal peptide; (c) a translation initiation codon that is flanked by a plant-preferred translation initiation context nucleotide sequence; and (d) an operably linked nucleotide sequence comprising a plant intron that is inserted upstream of the coding sequence. In some embodiments of this method, the biologically active recombinant polypeptide is secreted into the duckweed culture medium.
In some embodiments of the above methods, the signal peptide is selected from the group consisting of: (a) the human xcex1-2b-interferon signal sequence; (b) the Arabidopsis thaliana chitinase signal sequence; (c) the rice xcex1-amylase signal sequence; (d) the modified rice xcex1-amylase sequence; (e) a duckweed signal sequence; and (f) a signal sequence native to the biologically active recombinant polypeptide. In one embodiment of the method, the signal peptide is the rice xcex1-amylase signal peptide whose sequence is set forth in SEQ ID NO:3.
In some embodiments of the above methods, the duckweed-preferred codons are Lemna-preferred codons. In further embodiments, the duckweed-preferred codons are Lemna gibba-preferred codons or Lemna minor-preferred codons. In further embodiments, at least one coding sequence selected from the coding sequence for the biologically active recombinant polypeptide and the coding sequence for the signal peptide comprises between 70-100% Lemna gibba-preferred codons or Lemna minor-preferred codons.
In other embodiments, the invention encompasses a method of producing a biologically active recombinant polypeptide, comprising the steps of: (a) culturing a duckweed plant culture or a duckweed nodule culture, wherein said duckweed plant culture or said duckweed nodule culture is stably transformed to express said biologically active recombinant polypeptide, and wherein said biologically active recombinant polypeptide is encoded by a nucleotide sequence that has been modified for enhanced expression in duckweed, and (b) collecting said biologically active polypeptide from said duckweed plant culture or said duckweed nodule culture. In some embodiments of this method, the nucleotide sequence that has been modified for enhanced expression in duckweed has at least one attribute selected from the group consisting of: (a) duckweed-preferred codons in the coding sequence for said biologically active recombinant polypeptide; (b) a translation initiation codon that is flanked by a plant-preferred translation initiation context nucleotide sequence; and (c) an operably linked nucleotide sequence comprising a plant intron that is inserted upstream of the coding sequence.
In some embodiments of this method, the duckweed-preferred codons are Lemna-preferred codons. In further embodiments, the duckweed-preferred codons are Lemna gibba-preferred codons or Lemna minor-preferred codons. In further embodiments, the coding sequence comprises between 70-100% Lemna gibba-preferred codons or Lemna minor-preferred codons.
In some embodiments of the above methods, the plant-preferred translation initiation context nucleotide sequence consists of the nucleotide sequence xe2x80x9cACCxe2x80x9d or xe2x80x9cACAxe2x80x9d, wherein said context is positioned immediately adjacent to the 5xe2x80x2 end of the translation initiation codon.
In some embodiments of the above methods, the operably linked nucleotide sequence comprising said plant intron is the sequence set forth in SEQ ID NO:1.
In some embodiments of any of the above methods, the duckweed frond culture or duckweed nodule culture expresses and assembles all of the subunits of a biologically active multimeric protein. In further embodiments, the multimeric protein is selected from the group consisting of collagen, hemoglobin, P450 oxidase, and a monoclonal antibody.
In some embodiments of any of the above methods, the biologically active recombinant polypeptide is a mammalian polypeptide. In further embodiments, the mammalian polypeptide is a therapeutic polypeptide. In some embodiments, the mammalian polypeptide is selected from the group consisting of: insulin, growth hormone, xcex1-interferon, xcex2-interferon, xcex2-glucocerebrosidase, xcex2-glucoronidase, retinoblastoma protein, p53 protein, angiostatin, leptin, monoclonal antibodies, cytokines, receptors, human vaccines, animal vaccines, plant polypeptides, and serum albumin.
In some embodiments of any of the above methods, the biologically active recombinant polypeptide is xcex1-2b-interferon. In further embodiments, the xcex1-2b-interferon is human xcex1-2b-interferon. In further embodiments, the human xcex1-2b-interferon has the amino acid sequence set forth in SEQ ID NO:4 or SEQ ID NO:5.
In some embodiments of any of the above methods, the biologically active recombinant polypeptide is a biologically active variant of xcex1-2b-interferon, wherein said biologically active variant has at least 80% sequence identity with SEQ ID NO:4 or SEQ ID NO:5.
In some embodiments of any of the above methods, the biologically active recombinant polypeptide is an enzyme.
In other embodiments, the invention encompasses the stably transformed duckweed plant culture or duckweed nodule culture of any of the above methods. In further embodiments, the stably transformed duckweed plant culture or duckweed nodule culture is selected from the group consisting of the genus Spirodela, genus Wolffia, genus Wolfiella, and genus Lemna. In further embodiments, the stably transformed duckweed plant culture or duckweed nodule culture is selected from the group consisting of Lemna minor, Lemna miniscula, Lemna aequinoctialis, and Lemna gibba. 
In other embodiments, the invention encompasses a nucleic acid molecule comprising a nucleotide sequence encoding an amino acid sequence selected from the group consisting of: (a) the amino acid sequence set forth in SEQ ID NO:4; (b) the amino acid sequence set forth in SEQ ID NO:5; (c) the amino acid sequence of a biologically active variant of the amino acid sequence shown in SEQ ID NO:4, wherein said biologically active variant has at least about 80% sequence identity with the amino acid sequence set forth in SEQ ID NO:4; and (d) the amino acid sequence of a biologically active variant of the amino acid sequence shown in SEQ ID NO:5, wherein said biologically active variant has at least about 80% sequence identity with the amino acid sequence set forth in SEQ ID NO:5; wherein the nucleotide sequence comprises duckweed-preferred codons. In further embodiments, the nucleotide sequence is the nucleotide sequence set forth in SEQ ID NO:2.
In other embodiments, the invention encompasses a nucleic acid molecule comprising a nucleotide sequence encoding a signal peptide selected from the group consisting of: (a) the rice xcex1-amylase signal peptide amino acid sequence set forth in SEQ ID NO:6; and (b) the modified rice-amylase signal peptide amino acid sequence set forth in SEQ ID NO:7; wherein the nucleotide sequence comprises duckweed-preferred codons. In further embodiments, the nucleotide sequence is the nucleotide sequence set forth in SEQ ID NO:3.
In other embodiments, the invention encompasses a nucleic acid molecule for the expression and secretion of human xcex1-2b-interferon in duckweed comprising the signal peptide-encoding nucleotide sequence given in SEQ ID NO:3, and the mature human xcex1-2b-interferon-encoding nucleotide sequence given in SEQ ID NO:5, wherein the signal peptide-encoding nucleotide sequence and said mature human xcex1-2b-interferon-encoding nucleotide sequence are operably linked. In further embodiments, the nucleic acid molecule additionally comprises the intron-comprising nucleotide sequence given in SEQ ID NO:1, wherein said intron-comprising nucleotide sequence is operably linked to said signal peptide-encoding nucleotide sequence and said mature human xcex1-2b-interferon-encoding nucleotide sequence.
These and other aspects of the present invention are disclosed in more detail in the description of the invention given below.